Methods and compositions for evaluating graft survival in a solid organ transplant recipient

ABSTRACT

Methods are provided for evaluating a subject for graft survival, e.g., in terms of predicting graft survival, identifying the presence of a deleterious graft condition, such as CAN and DT, identifying the severity and class of acute rejection, etc, in a subject are provided. In practicing the subject methods, the expression of at least one gene in a sample from the subject, e.g., a blood or biopsy sample, is assayed, e.g., at the nucleic acid and/or protein level, to evaluate the subject. Also provided are compositions, systems and kits that find use in practicing the subject methods. The methods and compositions find use in a variety of applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

Notice: More than one reissue application has been filed for the reissueof U.S. Pat. No. 7,741,038. The reissue applications are applicationSer. No. 13/529,768 (the present application), and Ser. No. 13/943,626,filed Jul. 16, 2013 (a continuation reissue application).

Pursuant to 35 U.S.C. §119 (e), this application This application is aReissue of U.S. Pat. No. 7,741,038, which patent issued on Jun. 22,2010, and which patent claims priority to the filing date of U.S.Provisional Patent Application Ser. No. 60/662,083 filed on Mar. 14,2005; the disclosure of which application is herein incorporated byreference.

BACKGROUND

Transplantation of a graft organ or tissue from a donor to a hostpatient is a feature of certain medical procedures and treatmentprotocols. Despite efforts to avoid graft rejection through host-donortissue type matching, in transplantation procedures where a donor organis introduced into a host, immunosuppressive therapy is generallyrequired to the maintain viability of the donor organ in the host.

After an organ has been transplanted into the patient, the patient'simmune system is suppressed to prevent rejection of the new organ.Despite the wide use of immunosuppressive therapy, organ transplantrejection can occur.

Organ transplant rejection comprises three separate categories:hyperacute, acute and chronic. Hyperacute rejection is characterized byrapid thrombotic occlusion of the graft vasculature within minutes tohours after organ transplantation. Hyperacute rejection is mediated inlarge part by preexisting antibodies that bind to the epithelium andactivate the complement cascade. Complement activation results inendothelial cell damage and subsequent exposure of the basementmembrane, resulting in the activation of platelets, leading tothrombosis and vascular occlusion. As the field of transplantation hasmatured, hyperacute rejection has become less common due to bloodantigen and MHC molecule matching between the donor organ and therecipient.

Acute rejection is sub-classified into acute vascular rejection andacute cellular rejection. Acute vascular rejection is characterized bynecrosis of individual cells in the graft blood vessels. The process issimilar to that of hyperacute rejection, but onset is often slower,within one week of rejection, and a T cell component may be involved.Acute vascular rejection is initiated by a response to alloantigenspresent on the vascular endothelial cells of the donor organ, resultingin the release of a cytokine cascade, inflammation, and eventualnecrosis. Acute cellular rejection is often characterized by necrosis ofthe essential or parenchymal cells of the transplanted organ caused bythe infiltration of host T lymphocytes and macrophages. The lymphocytesinvolved are usually cytotoxic T lymphocytes (CTL) and macrophages, bothresulting in lysis of targeted cells. The CTLs are usually specific forgraft alloantigens displayed in the context of MHC class I molecules.

Chronic rejection is the major cause of allograft loss and ischaracterized by fibrosis and loss of normal organ structures. Fibrosismay be the result of wound healing following the cellular necrosis ofacute rejection, or may occur independently and without prior acuterejection. In addition, chronic rejection may lead to vascularocclusions thought to stem from a delayed type hypersensitivity responseto alloantigens present on the transplanted organ. These alloantigensstimulate lymphocytes to secrete cytokines which attract macrophages andother effector cells eventually leading to an arteriosclerosis-likeblockage.

In many cases, chronic graft injury or rejection (CR) is largely due tocalcineurin-inhibitor drug nephrotoxicity (DT) and chronic allograftnephropathy (CAN), two conditions which may result in loss of graftfunction and early graft loss, premature to the life expectancy of therecipient. The incidence of chronic graft loss has remained unchangedover the last decade.

A biopsy is the only current gold standard for CAN and DT diagnosis. Asboth conditions are progressive post-transplantation, multiple graftprotocol biopsies are required. However, the invasiveness of biopsyprocedures is a limitation to this form of monitoring. In addition,variability of biopsy sampling and pathology analysis (2) adds aconfounder to the differential diagnosis of these 2 conditions—theresult of either too much drug (DT) vs. too little/inappropriate drugs(CAN)—with a common outcome of chronic fibrotic injury from differingmechanisms (non-immune vs. immune).

There is currently no method available to detect or to monitor futuregraft loss at the time of transplantation or acute rejection (AR)episodes. AR is a risk factor both for eventual graft loss, delayedrecovery of graft function and even chronic rejection. Non-invasivemonitoring methods for AR stratification, CR, DT and developing orestablished tolerance is currently not available, but would be veryvaluable, as the transplant biopsy, though the current gold standard,fails to stratify or prognosticate AR, differentiate CR clearly from DTor diagnose tolerance.

Accordingly, of interest would be the ability to evaluate likelihood ofgraft survival in a transplant recipient, e.g., following an AR episode,such that treatment protocols for transplant patients may be customized.

SUMMARY OF THE INVENTION

Methods are provided for evaluating a subject for graft survival, e.g.,in terms of predicting graft survival, identifying the presence of adeleterious graft condition, such as CAN and DT, identifying theseverity and class of acute rejection, etc, in a subject are provided.In practicing the subject methods, the expression of at least one genein a sample from the subject, e.g., a blood or biopsy sample, isassayed, e.g., at the nucleic acid and/or protein level, to evaluate thesubject. Also provided are compositions, systems and kits that find usein practicing the subject methods.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

“Acute rejection or AR” is the rejection by the immune system of atissue transplant recipient when the transplanted tissue isimmunologically foreign. Acute rejection is characterized byinfiltration of the transplanted tissue by immune cells of therecipient, which carry out their effector function and destroy thetransplanted tissue. The onset of acute rejection is rapid and generallyoccurs in humans within a few weeks after transplant surgery. Generally,acute rejection can be inhibited or suppressed with immunosuppressivedrugs such as rapamycin, cyclosporin A, anti-CD40L monoclonal antibodyand the like.

“Chronic transplant rejection or CR” generally occurs in humans withinseveral months to years after engraftment, even in the presence ofsuccessful immunosuppression of acute rejection. Fibrosis is a commonfactor in chronic rejection of all types of organ transplants. Chronicrejection can typically be described by a range of specific disordersthat are characteristic of the particular organ. For example, in lungtransplants, such disorders include fibroproliferative destruction ofthe airway (bronchiolitis obliterans); in heart transplants ortransplants of cardiac tissue, such as valve replacements, suchdisorders include fibrotic atherosclerosis; in kidney transplants, suchdisorders include, obstructive nephropathy, nephrosclerorsis,tubulointerstitial nephropathy; and in liver transplants, such disordersinclude disappearing bile duct syndrome. Chronic rejection can also becharacterized by ischemic insult, denervation of the transplantedtissue, hyperlipidemia and hypertension associated withimmunosuppressive drugs.

The term “transplant rejection” encompasses both acute and chronictransplant rejection.

The term “stringent assay conditions” as used herein refers toconditions that are compatible to produce binding pairs of nucleicacids, e.g., surface bound and solution phase nucleic acids, ofsufficient complementarity to provide for the desired level ofspecificity in the assay while being less compatible to the formation ofbinding pairs between binding members of insufficient complementarity toprovide for the desired specificity. Stringent assay conditions are thesummation or combination (totality) of both hybridization and washconditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization (e.g., as inarray, Southern or Northern hybridizations) are sequence dependent, andare different under different experimental parameters. Stringenthybridization conditions that can be used to identify nucleic acidswithin the scope of the invention can include, e.g., hybridization in abuffer comprising 50% formamide, 5×SSC, and 1% SDS at 42° C., orhybridization in a buffer comprising 5×SSC and 1% SDS at 65° C., bothwith a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplary stringenthybridization conditions can also include hybridization in a buffer of40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in 1×SSC at45° C. Alternatively, hybridization to filter-bound DNA in 0.5 M NaHPO₄,7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. can be employed. Yet additional stringenthybridization conditions include hybridization at 60° C. or higher and3×SSC (450 mM sodium chloride/45 mM sodium citrate) or incubation at 42°C. in a solution containing 30% formamide, 1M NaCl, 0.5% sodiumsarcosine, 50 mM MES, pH 6.5. Those of ordinary skill will readilyrecognize that alternative but comparable hybridization and washconditions can be utilized to provide conditions of similar stringency.

In certain embodiments, the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid isspecifically hybridized to a surface bound nucleic acid. Wash conditionsused to identify nucleic acids may include, e.g.: a salt concentrationof about 0.02 molar at pH 7 and a temperature of at least about 50° C.or about 55° C. to about 60° C.; or, a salt concentration of about 0.15M NaCl at 72° C. for about 15 minutes; or, a salt concentration of about0.2×SSC at a temperature of at least about 50° C. or about 55° C. toabout 60° C. for about 15 to about 20 minutes; or, the hybridizationcomplex is washed twice with a solution with a salt concentration ofabout 2×SSC containing 0.1% SDS at room temperature for 15 minutes andthen washed twice by 0.1×SSC containing 0.1% SDS at 68° C. for 15minutes; or, equivalent conditions. Stringent conditions for washing canalso be, e.g., 0.2×SSC/0.1% SDS at 42° C.

A specific example of stringent assay conditions is rotatinghybridization at 65° C. in a salt based hybridization buffer with atotal monovalent cation concentration of 1.5 M (e.g., as described inU.S. patent application Ser. No. 09/655,482 filed on Sep. 5, 2000, thedisclosure of which is herein incorporated by reference) followed bywashes of 0.5×SSC and 0.1×SSC at room temperature.

Stringent assay conditions are hybridization conditions that are atleast as stringent as the above representative conditions, where a givenset of conditions are considered to be at least as stringent ifsubstantially no additional binding complexes that lack sufficientcomplementarity to provide for the desired specificity are produced inthe given set of conditions as compared to the above specificconditions, where by “substantially no more” is meant less than about5-fold more, typically less than about 3-fold more. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide,including exon and (optionally) intron sequences. The term “intron”refers to a DNA sequence present in a given gene that is not translatedinto protein and is generally found between exons in a DNA molecule. Inaddition, a gene may optionally include its natural promoter (i.e., thepromoter with which the exons and introns of the gene are operablylinked in a non-recombinant cell, i.e., a naturally occurring cell), andassociated regulatory sequences, and may or may not have sequencesupstream of the AUG start site, and may or may not include untranslatedleader sequences, signal sequences, downstream untranslated sequences,transcriptional start and stop sequences, polyadenylation signals,translational start and stop sequences, ribosome binding sites, and thelike.

A “protein coding sequence” or a sequence that “encodes” a particularpolypeptide or peptide, is a nucleic acid sequence that is transcribed(in the case of DNA) and is translated (in the case of mRNA) into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from viral, procaryotic oreukaryotic mRNA, genomic DNA sequences from viral, procaryotic oreukaryotic DNA, and even synthetic DNA sequences. A transcriptiontermination sequence may be located 3′ to the coding sequence.

The terms “reference” and “control” are used interchangebly to refer toa known value or set of known values against which an observed value maybe compared. As used herein, known means that the value represents anunderstood parameter, e.g., a level of expression of a marker gene in agraft survival or loss phenotype.

The term “nucleic acid” includes DNA, RNA (double-stranded or singlestranded), analogs (e.g., PNA or LNA molecules) and derivatives thereof.The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides. The terms “deoxyribonucleic acid” and “DNA”as used herein mean a polymer composed of deoxyribonucleotides. The term“mRNA” means messenger RNA. An “oligonucleotide” generally refers to anucleotide multimer of about 10 to 100 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having any number ofnucleotides.

The terms “protein” and “polypeptide” used in this application areinterchangeable. “Polypeptide” refers to a polymer of amino acids (aminoacid sequence) and does not refer to a specific length of the molecule.Thus peptides and oligopeptides are included within the definition ofpolypeptide. This term does also refer to or include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylation and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid, polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring.

The term “assessing” and “evaluating” are used interchangeably to referto any form of measurement, and includes determining if an element ispresent or not. The terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Predictive Analysis of Microarrays (PAM) using a set of 3,170differentially expressed genes identifies the 33 classifiers withsimilar power (FIG. 1A). The PAM classification scores grouped thesamples with 100% concordance to assigned classes and reported scoresare aligned with the clustered samples (FIG. 1B).

FIG. 2. Kaplan-Meier survival analysis for graft loss (red) and no-loss(blue). The genes include ICAM5 (FIG. 2A; p=0.007), IL6R (FIG. 2B;p=0.003), STAT1 (FIG. 2C; p=0.036), and STAT6 (FIG. 2D (p=0.020).

FIG. 3. Kaplan-Meier survival curves for 8 genes from whole bloodsamples that are predictive of graft loss. Genes include AHSA2 (FIG.3A), IGHG1 (FIG. 3B), IFNAR2 (FIG. 3C), IGKC (FIG. 3D), HIST1H2BC (FIG.3E), IL1R2 (FIG. 3F), MAPK1 (FIG. 3G), and MAPK9 (FIG. 3H).

FIG. 4. Demonstrates that gene expression is generallyuniform/consistent across the full clinical groups analyzed as the geneexpression levels segregate well within patient groups.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for evaluating a subject for graft function, e.g.,in terms of predicting graft survival, identifying the presence of adeleterious graft condition, such as CAN and DT, identifying theseverity and class of acute rejection, etc, in a subject are provided.In practicing the subject methods, the expression of at least one genein a sample from the subject, e.g., a blood or biopsy sample, isassayed, e.g., at the nucleic acid and/or protein level, to evaluate thesubject. Also provided are compositions, systems and kits that find usein practicing the subject methods. The methods and compositions find usein a variety of applications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, the subject invention is directed to methods ofevaluating graft function in a subject, as well as reagents and kits foruse in practicing the subject methods. In further describing theinvention, the subject methods are described first, followed by a reviewof the reagents and kits for use in practicing the subject methods.

Methods of Evaluating Graft Function

As reviewed above, the subject invention provides methods for evaluatinga subject for graft survival. The methods provide for evaluating asubject for graft survival in terms of a number of different factors. Incertain embodiments, the factor evaluated is a basic prediction of graftsurvival. In certain embodiments, the factor evaluated is the presenceof a deleterious graft condition, such as CAN and DT. In certainembodiments, the factor identified is the severity and/or class of acuterejection, where these embodiments are distinguished from methods thatjust identify the presence of acute rejection, since one is furtherdetermining the severity and/or class of acute rejection, and thereforean aspect of graft survival

As such, certain embodiments of the invention provide methods ofevaluating, e.g., in terms of predicting, graft survival in a subjectcomprising a graft. As such, the subject invention provides methods ofevaluating whether a graft in a transplant patient or subject willsurvive or be lost. In certain embodiments, the methods may be viewed asmethods of determining whether a transplant subject has a graft survivalphenotype, i.e., a phenotype in which the graft will survive. A graftsurvival phenotype is a phenotype characterized by the presence oflong-term graft survival. By “long-term” graft survival is meant graftsurvival for at least about 5 years beyond current sampling, despite theoccurrence of one or more prior episodes of AR. In certain embodiments,graft survival is determined for patients in which at least one episodeof acute rejection (AR) has occurred. As such, these embodiments aremethods of determining or predicting graft survival following AR. Graftsurvival is determined or predicted in certain embodiments in thecontext of transplant therapy, e.g., immunosuppressive therapy, whereimmunosuppressive therapies are known in the art. In yet otherembodiments, methods of distinguishing being organ rejection diseaseconditions, such as CAN and DT, are provided. In yet other embodiments,methods of determining the class and/or severity of acute rejection (andnot just the presence thereof are provided.

As in known in the transplantation field, the graft organ, tissue orcell(s) may be allogeneic or xenogeneic, such that the grafts may beallografts or xenografts. Organs and tissues of interest include, butare not limited to: skin, heart, kidney, liver, bone marrow, and otherorgans.

In practicing the subject methods, a subject or patient sample, e.g.,cells or collections thereof, e.g., tissues, is assayed to evaluategraft survival in the host, e.g., whether the graft will survive in thehost from which the assayed sample was obtained. Accordingly, the firststep of the subject methods is to obtain a suitable sample from thesubject or patient of interest, i.e., a patient having at least onegraft, e.g., allograft.

The sample is derived from any initial suitable source, where samplesources of interest include, but are not limited to, many differentphysiological sources, e.g., CSF, urine, saliva, tears, tissue derivedsamples, e.g., homogenates (such as biopsy samples of the transplantedtissue or organ (including, but not limited to kidney, heart, lungbiopsies), and blood or derivatives thereof.

In certain embodiments, a suitable initial source for the patient sampleis blood. As such, the sample employed in the subject assays of theseembodiments is generally a blood-derived sample. The blood derivedsample may be derived from whole blood or a fraction thereof, e.g.,serum, plasma, etc., where in certain embodiments the sample is derivedfrom blood cells harvested from whole blood. Of particular interest as asample source are peripheral blood lymphocytes (PBL). Any convenientprotocol for obtaining such samples may be employed, where suitableprotocols are well known in the art and a representative protocol isreported in the Experimental Section, below.

In practicing the subject methods, the sample is assayed to obtain anexpression evaluation, e.g., expression profile, for one or more genes,where the term expression profile is used broadly to include a genomicexpression profile, e.g., an expression profile of nucleic acidtranscripts, e.g., mRNAs, of the one or more genes of interest, or aproteomic expression profile, e.g., an expression profile of one or moredifferent proteins, where the proteins/polypeptides are expressionproducts of the one or more genes of interest. As such, in certainembodiments the expression of only one gene is evaluated. In yet otherembodiments, the expression of two or more, e.g., about 5 or more, about10 or more, about 15 or more, about 25 or more, about 50 or more, about100 or more, about 200 or more, etc., genes is evaluated. Accordingly,in the subject methods, the expression of at least one gene in a sampleis evaluated. In certain embodiments, the evaluation that is made may beviewed as an evaluation of the transcriptosome, as that term is employedin the art. See e.g., Gomes et al., Blood (2001 Jul. 1) 98(1): 93-9.

In generating the expression profile, in certain embodiments a sample isassayed to generate an expression profile that includes expression datafor at least one gene/protein, usually a plurality of genes/proteins,where by plurality is meant at least two different genes/proteins, andoften at least about 5, typically at least about 10 and more usually atleast about 20 different genes/proteins or more, such as 50 or more, 100or more, etc.

In the broadest sense, the expression evaluation may be qualitative orquantitative. As such, where detection is qualitative, the methodsprovide a reading or evaluation, e.g., assessment, of whether or not thetarget analyte, e.g., nucleic acid or expression product, is present inthe sample being assayed. In yet other embodiments, the methods providea quantitative detection of whether the target analyte is present in thesample being assayed, i.e., an evaluation or assessment of the actualamount or relative abundance of the target analyte, e.g., nucleic acidin the sample being assayed. In such embodiments, the quantitativedetection may be absolute or, if the method is a method of detecting twoor more different analytes, e.g., target nucleic acids, in a sample,relative. As such, the term “quantifying” when used in the context ofquantifying a target analyte, e.g., nucleic acid(s), in a sample canrefer to absolute or to relative quantification. Absolute quantificationmay be accomplished by inclusion of known concentration(s) of one ormore control analytes and referencing the detected level of the targetanalyte with the known control analytes (e.g., through generation of astandard curve). Alternatively, relative quantification can beaccomplished by comparison of detected levels or amounts between two ormore different target analytes to provide a relative quantification ofeach of the two or more different analytes, e.g., relative to eachother.

Genes/proteins of interest are graft survival/loss indicative genes,i.e., genes/proteins that are differentially expressed or present atdifferent levels in graft survival and graft loss individuals (morespecifically, individuals in which graft loss will occur vs. individualsin which a graft will survive). Representative genes/proteins ofinterest in certain embodiments include, but are not limited to, thegenes/proteins provided in Tables 1 and 2. (Note that for Tables 1 and2, the exact sequence of the clone identified in the table can bedetermined through the NCBI Entrez nucleotide database located at thewebsite produced by placing “http://www.” before:“ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&db=nucleotide” in thenavigation window of a web browser (e.g., Netscape); the sequence for aspecific clone is then obtained by entering the clone ID in quotes asthe search term).

TABLE 1 Genes of known function in whole blood predictive of graft lossfollowing acute rejection Rank Clone Symbol Gene UnigeneID 1 IMAGE:214006 HIST1H2BC Histone 1, H2bc Hs.356901 2 IMAGE: 826131 IGHG3 Igheavy constant gamma 3 Hs.413826 3 IMAGE: 626318 UBN1 Ubinuclein 1Hs.21479 4 IMAGE: 511387 GLG1 Golgi apparatus protein 1 Hs.78979 5IMAGE: 810057 CSDA Cold shock domain protein A Hs.221889 6 IMAGE: 283919HIST1H2AC Histone 1, H2ac Hs.28777 7 IMAGE: 453710 PLEK2 Pleckstrin 2Hs.170473 8 IMAGE: 840821 SSR4 Signal sequence receptor, delta Hs.4092239 IMAGE: 70201 MSCP Mitochondrial solute carrier Hs.283716 10 IMAGE:66686 RPL10 Ribosomal protein L10 Hs.77091 11 IMAGE: 1306420 AHSA2Activator of heat shock ATPase Hs.122440 12 IMAGE: 2578221 UBB UbiquitinB Hs.356190 13 IMAGE: 811062 CGI-69 CGI-69 protein Hs.237924 14 IMAGE:1272566 TNFRSF10D TNF receptor superfamily 10d Hs.129844 15 IMAGE:1240649 RPL10 Ribosomal protein L10 Hs.77091 16 IMAGE: 85224 RBM25 RNAbinding motif protein 25 Hs.197184 17 IMAGE: 2114004 HIST1H3D Histone 1,H3d Hs.239458 18 IMAGE: 789091 HIST1H2AC Histone 1, H2ac Hs.28777 19IMAGE: 591025 JMJD3 Jumonji domain containing 3 Hs.103915 20 IMAGE:1354406 SSR4 Signal sequence receptor, delta Hs.409223 21 IMAGE: 812276SNCA Synuclein Hs.76930 22 IMAGE: 344720 GYPC Glycophorin C Hs.81994 23IMAGE: 683899 JMJD3 Jumonji domain containing 3 Hs.103915 24 IMAGE:825006 CYorf15A Chromosome Y ORF Hs.171857 25 IMAGE: 1492412 UBA52Ubiquitin A-52 fusion product 1 Hs.5308 26 IMAGE: 854079 ACTN1 Actinin,alpha 1 Hs.119000 27 IMAGE: 366884 IFNAR2 Interferon (a- B- and o)receptor 2 Hs.86958 28 IMAGE: 812967 TM4SF9 Transmembrane 4 superfamilyHs.8037 29 IMAGE: 207794 NFE2 Erythroid nuclear factor Hs.75643 30IMAGE: 359835 SAT Spermidine N1-acetyltransferase Hs.28491 31 IMAGE:565849 KLHL12 Kelch-like 12 (Drosophila) Hs.3826 32 IMAGE: 256260 RFC3Replication factor C activator Hs.115474 33 IMAGE: 191826 MSCPMitochondrial solute carrier protein Hs.283716 34 IMAGE: 202242 MIFMacrophage migration inhibitor Hs.407995 35 IMAGE: 323506 MAPK1Mitogen-activated protein kinase 1 Hs.324473 36 IMAGE: 1286850 MMEMembrane metallo-endopeptidase Hs.259047 37 IMAGE: 129725 RBPSUHRecombining binding protein Hs.347340 38 IMAGE: 882522 ASSArgininosuccinate synthetase Hs.160786 39 IMAGE: 2129439 UBE2BUbiquitin-conjugating enzyme E2B Hs.385986 40 IMAGE: 1687138 HIST1H2AMHistone 1, H2am Hs.134999 41 IMAGE: 209655 TGFBR3 TGFb receptor IIIHs.342874 42 IMAGE: 75254 CSRP2 Cysteine and glycine-rich protein 2Hs.10526 43 IMAGE: 1715851 HBG2 Hemoglobin, gamma G Hs.302145 44 IMAGE:155467 SLC9A3R2 Solute carrier family 9 Hs.440896 45 IMAGE: 561743PPP1R1A Protein phosphatase 1 Hs.435238 46 IMAGE: 565075 STC1Stanniocalcin 1 Hs.25590 47 IMAGE: 1541958 POU2AF1 POU domainassociating factor Hs.2407 48 IMAGE: 324122 ESM1 Endothelialcell-specific molecule 1 Hs.129944 49 IMAGE: 80338 SELENBP1 Seleniumbinding protein 1 Hs.334841 50 IMAGE: 1472754 COX6B1 Cytochrome coxidase (ubiquitous) Hs.431668 51 IMAGE: 233583 IL1R2 Interleukin 1receptor, type II Hs.25333 52 IMAGE: 490060 RNF159 Ring finger protein(C3HC4 type) Hs.246914 53 IMAGE: 1185475 ABCC5 ATP-binding cassette CHs.22010 54 IMAGE: 120551 LPIN2 Lipin 2 Hs.437425 55 IMAGE: 162772 EGR1Early growth response 1 Hs.326035 56 IMAGE: 322029 MAPK9Mitogen-activated protein kinase 9 Hs.348446 57 IMAGE: 1305158 KIAA1219KIAA1219 protein Hs.348929 58 IMAGE: 2505604 SCYE1 Endothelialmonocyte-activating) Hs.105656 59 IMAGE: 1240813 IGKC Immunoglobulinkappa constant Hs.377975 60 IMAGE: 257637 RRBP1 Ribosome binding protein1 homolog Hs.98614 61 IMAGE: 381522 PP1057 Hypothetical protein PP1057Hs.108557 62 IMAGE: 455123 MTSS1 Metastasis suppressor 1 Hs.77694

TABLE 2 Genes of known function in renal biopsies whole blood predictiveof graft loss following acute rejection. Unigene Rank Clone Symbol GeneID 1 IMAGE: 2134209 ZNF41 Zinc finger protein 41 Hs.143700 2 IMAGE:1241524 TCL1A T-cell leukemia/lymphoma 1A Hs.2484 3 IMAGE: 704915 TAP1Transporter 1 (MDR/TAP) Hs.352018 4 IMAGE: 267600 STAT6 Interleukin-4induced STAT6 Hs.437475 5 IMAGE: 26599 STAT1 Interleukin-4 induced STAT1Hs.21486 6 IMAGE: 210405 PSME2 Proteasome activator Hs.434081 7 IMAGE:1240661 PSMB9 Proteasome beta type, 9 Hs.381081 8 IMAGE: 705046 PMLPromyelocytic leukemia Hs.89633 9 IMAGE: 824340 NCF1 Neutrophilcytosolic factor 1 Hs.1583 10 IMAGE: 753313 LAPTM5 Lysosomal-associatedprotein-5 Hs.436200 11 IMAGE: 1351990 ISG20 Interferon stimulated gene20 kDa Hs.105434 12 IMAGE: 1672498 IGLV@ Ig lambda variable groupHs.449601 13 IMAGE: 1240590 IGLC2 Ig lambda constant 2 Hs.405944 14IMAGE: 1240813 IGKC Ig kappa constant Hs.377975 15 IMAGE: 1604703 HLA-FMHC complex, class I, F Hs.411958 16 IMAGE: 2448698 HLA-DRB6 MHC, classII, DR beta 6 (pseudogene) Hs.534338 17 IMAGE: 461769 HLA-DRB5 MHCcomplex, class II, DR beta 5 Hs.308026 18 IMAGE: 1241341 HLA-DRB3 MHCcomplex, class II, DR beta 3 Hs.520049 19 IMAGE: 1241211 HLA-DPB1 MHCcomplex, class II, DP beta 1 Hs.368409 20 IMAGE: 203527 HLA-A MHCcomplex, class I, A Hs.181244 21 IMAGE: 853906 HCG4P6 HLA complex group4 pseudogene 6 Hs.512759 22 IMAGE: 841008 GBP1 Guanylate binding 1,interferon-inducible Hs.62661 23 IMAGE: 277522 DAF Decay acceleratingfactor complement (CD55) Hs.408864 24 IMAGE: 269295 CD83 CD83 antigen(Activated B lymphocytes) Hs.444310 25 IMAGE: 276727 CD69 CD69 antigen(early T-cell activation antigen) Hs.82401 26 IMAGE: 200720 CD38 CD38antigen (p45) Hs.174944 27 IMAGE: 2000918 CAS1 O-acetyltransferaseHs.324725 28 IMAGE: 67042 APOM Apolipoprotein M Hs.247323 29 IMAGE:488143 IGHM Immunoglobulin heavy locus Hs.103995 30 IMAGE: 207718 TASSIg light chain variable region Hs.449578

In certain embodiments, at least one of the genes/proteins in theprepared expression profile is a graft survival/rejection indicativegene from Tables 1 and/or 2, where the expression profile may includeexpression data for 5, 10, 20, 50, 75 or more of, including all of, thegenes/proteins listed in Tables 1 and/or 2. The number of differentgenes/proteins whose expression and/or quantity data, i.e., presence orabsence of expression, as well as expression/quantity level, that areincluded in the expression profile that is generated may vary, but maybe at least 2, and in certain embodiments ranges from 2 to about 100 ormore, sometimes from 3 to about 75 or more, including from about 4 toabout 70 or more.

In certain embodiments, additional genes beyond those listed in Tables 1and/or 2, may be assayed, such as genes whose expression pattern can beused to evaluate additional transplant characteristics, including butnot limited to: acute rejection (e.g., the genes identified as AR inTable 3, below); chronic allograft injury (chronic rejection) in blood(e.g., the genes identified as CR in Table 3, below); immunosuppressivedrug toxicity or adverse side effects including drug-inducedhypertension (e.g., the genes identified as DT in Table 3, below); ageor body mass index associated genes that correlate with renal pathologyor account for differences in recipient age-related graft acceptance(e.g., the genes identified as BMI in Table 3, below); immune tolerancemarkers in whole blood (e.g., the genes identified as TOL in Table 3,below); genes found in literature surveys with immune modulatory rolesthat may play a role in transplant outcomes (e.g., the genes identifiedas Lit. in Table 3, below); as well as other array assay functionrelated genes, e.g., for assessing sample quality (3′- to 5′-bias inprobe location), sampling error in biopsy-based studies, cell surfacemarkers, and normalizing genes for calibrating hybridization results(see e.g., the genes identified as Contr. in Table 3, below); and thelike.

A representative collection of genes that includes not only graftsurvival/rejection genes of Tables 1 and 2 above, but also additionalgraft characterizing genes (e.g., specific for DT, CAN, and immunetolerance) is in Table 3.

TABLE 3 Genes of known function of prognostic value compiled for acustom transplantation chip (TxChip VI). Symbol Name mRNA Tissue StudyACOX1 Acyl-Coenzyme A oxidase 1, palmitoyl NM_004035 Blood AR ADD3Adducin 3 (gamma) NM_016824 Blood AR ADM Adrenomedullin NM_001124 BloodAR AHR Aryl hydrocarbon receptor NM_001621 Blood AR ATP1A1 ATPase,Na+/K+ transporting, alpha 1 NM_000701 Blood AR BUB1B BUB1 buddinguninhibited by benzimidazoles NM_001211 Blood AR CASP8 Caspase 8,apoptosis-related cysteine protease NM_001228 Blood AR CASP8AP2 CASP8associated protein 2 NM_012115 Blood AR CCNC Cyclin C NM_005190 Blood ARCD21 CD21 B-cell receptor for complement C3d0 Y00649 Blood AR CD69 CD69antigen (early T-cell activation antigen) NM_001781 Blood AR CD8A CD8antigen, alpha polypeptide (p32) NM_001768 Blood AR CDIPTPhosphatidylinositol synthase NM_145752 Blood AR COX6C Cytochrome coxidase subunit VIc NM_004374 Blood AR CSNK1A1 Casein kinase 1, alpha 1NM_001892 Blood AR DUSP1 Dual specificity phosphatase 1 NM_004417 BloodAR DUSP3 Dual specificity phosphatase 3 NM_004090 Blood AR EIF1AEukaryotic translation initiation factor 1A NM_001412 Blood AR EIF2S3Eukaryotic translation initiation factor 2 NM_001415 Blood AR GNLYGranulysin NM_006433 Blood AR GOLGIN-67 Golgin-67 XM_496064 Blood ARAHSA2 Activator of heat shock ATPase NM_152392 Blood AR HIST1H2BCHistone 1, H2bc NM_003526 Blood AR IFNAR2 Interferon (alpha, beta andomega) receptor 2 NM_000874 Blood AR IGHG1 Ig heavy constant gamma 1(G1m marker) AB067073 Blood AR IL1R2 Interleukin 1 receptor, type IINM_004633 Blood AR MAPK1 Mitogen-activated protein kinase 1 NM_002745Blood AR MIF Macrophage migration inhibitory factor NM_002415 Blood ARSCYE1 Endothelial monocyte-activating NM_004757 Blood AR TGFBR3 TGFbreceptor III (betaglycan) NM_003243 Blood AR TM4SF9 Transmembrane 4superfamily member 9 NM_005723 Blood AR IGHM Immunoglobulin heavyconstant mu X58529 Blood AR ISG20 Interferon stimulated gene 20 kDaNM_002201 Blood AR KIAA1014 FNBP4 formin binding protein 4 AB023231Blood AR LIV-1 SLC39A6 metal ion transporter NM_015359 Blood AR MAPKAPK5Mitogen-activated protein kinase NM_003668 Blood AR MDM4 p53 bindingprotein NM_002393 Blood AR MYT1 Myelin transcription factor 1 NM_004535Blood AR NAB1 EGR1 binding protein 1 NM_005966 Blood AR NFKB1 NFkBenhancer in B-cells 1 (p105) NM_003998 Blood AR PC4 RNA polymerase IItranscription cofactor 4 NM_006713 Blood AR PKM2 Pyruvate kinase, muscleNM_002654 Blood AR PTP4A1 Protein tyrosine phosphatase NM_003463 BloodAR RBL2 Retinoblastoma-like 2 (p130) NM_005611 Blood AR RBM3 RNA bindingmotif 3 (RNP1, RRM) NM_006743 Blood AR REL V-rel viral oncogene homologNM_002908 Blood AR RPL22 Ribosomal protein L22 NM_000983 Blood AR RPS24Ribosomal protein S24 NM_033022 Blood AR RPS27 Ribosomal protein S27NM_001030 Blood AR RPS4Y RPS4Y ribosomal protein S4 NM_001008 Blood ARSATB1 Special AT-rich sequence binding protein NM_002971 Blood AR SDS3Likely ortholog of mouse Sds3 NM_022491 Blood AR SSBP1 Single-strandedDNA binding protein 1 NM_003143 Blood AR SSI-3 SOCS3 suppressor ofcytokine signaling 3 NM_003955 Blood AR STK4 Serine/threonine kinase 4NM_006282 Blood AR TBRG1 Transforming growth factor beta regulator 1NM_032811 Blood AR TCF7 Transcription factor 7 (T-cell specific)NM_201633 Blood AR TOP2B Topoisomerase (DNA) II beta 180 kDa NM_001068Blood AR TRIM T-cell receptor interacting molecule NM_016388 Blood ARTRRAP Transcription domain-associated protein NM_003496 Blood AR UBA52Ubiquitin A-52-ribosomal protein fusion NM_003333 Blood AR UBB UbiquitinB NM_018955 Blood AR UBE2B Ubiquitin-conjugating enzyme E2B NM_003337Blood AR UBN1 Ubinuclein 1 NM_016936 Blood AR USP25 Ubiquitin specificprotease 25 NM_013396 Blood AR AIM1 Absent in melanoma 1 XM_166300Biopsy AR CD38 CD38 antigen (p45) NM_001775 Biopsy AR CDS1CDP-diacylglycerol synthase NM_001263 Biopsy AR CSF1R Feline sarcomaviral (v-fms) homolog NM_005211 Biopsy AR DR1 Down-regulator oftranscription 1 NM_001938 Biopsy AR FGL2 Fibrinogen-like 2 NM_006682Biopsy AR FLJ13612 Calcium binding protein AI635773 Biopsy AR HLA-A MHCclass I, A NM_002116 Biopsy AR HLA-B MHC class I, B NM_005514 Biopsy ARHLA-C MHC class I, C NM_002117 Biopsy AR HLA-DPA1 MHC class II, DP alpha1 NM_033554 Biopsy AR HLA-DRA MHC class II, DR alpha NM_019111 Biopsy ARIGKC Ig kappa constant AB064140 Blood AR TNFSF10 TNF superfamily, member10 NM_003810 Blood AR IGLJ3 IGLa Immunoglobulin lambda AI146764 BiopsyAR MYH10 Myosin, heavy polypeptide 10 NM_005964 Biopsy AR NKTR Naturalkiller-tumor recognition sequence NM_005385 Biopsy AR PAX8 Paired boxgene 8 NM_013951 Biopsy AR POLR2B Polymerase (RNA) II polypeptide BNM_000938 Biopsy AR RGN Regucalcin (senescence marker protein-30)NM_004683 Biopsy AR SCNN1A Sodium channel, nonvoltage-gated 1 alphaNM_001038 Biopsy AR SIM2 Single-minded homolog 2 NM_009586 Biopsy ARTACSTD2 Calcium signal transducer 2 NM_002353 Biopsy AR VCAM1 Vascularcell adhesion molecule 1 NM_001078 Biopsy AR YARS Tyrosyl-tRNAsynthetase NM_003680 Biopsy AR ZFP36L1 Zinc finger protein 36 NM_004926Biopsy AR HLA-DPB1 MHC, class II, DP beta 1 NM_002121 Biopsy AR HLA-DRB3MHC, class II, DR beta 4 NM_022555 Biopsy AR ACK1 Cdc42-associatedkinase 1 NM_005781 Biopsy AR HLA-F MHC, class I, F NM_018950 Biopsy ARICAM5 Intercellular adhesion molecule 5 NM_003259 Biopsy AR REG1ARegenerating islet-derived 1 alpha NM_002909 Biopsy AR GSTA2 GlutathioneS-transferase A2 NM_000846 Biopsy AR HLA-DRB5 MHC class II, DR beta 4NM_002125 Biopsy AR HLA-DQA1 MHC class II, DQ alpha 1 NM_002122 BiopsyAR HLA-DQB1 MHC class II, DQ beta 1 NM_002123 Biopsy AR RFXANKRegulatory factor X-associated ankyrin NM_003721 Biopsy AR STAT6Interleukin-4 induced STAT6 NM_003153 Biopsy AR TAP1 Transporter 1(MDR/TAP) NM_000593 Biopsy AR DAF Decay accelerating factor (CD55)NM_000574 Biopsy AR CD83 CD83 antigen (activated B lymphocytes)NM_004233 Biopsy AR STAT1 Interleukin-4 induced STAT1 NM_007315 BiopsyAR LTBR Lymphotoxin beta receptor NM_002342 Biopsy AR KCNJ1 Potassiuminwardly-rectifying channel NM_000220 Biopsy AR SLPI Secretory leukocyteprotease inhibitor NM_003064 Biopsy AR CD34 CD34 antigen NM_001773Biopsy AR HOXB5 Homeo box B5 NM_002147 Biopsy AR IL6R Interleukin 6receptor NM_181359 Biopsy AR DAPK1 Death-associated protein kinase 1NM_004938 Biopsy AR HOXD9 Homeo box D9 NM_014213 Biopsy AR TCF21Transcription factor 21 NM_003206 Biopsy AR MAL T-cell differentiationprotein NM_022438 Biopsy AR MAF V-maf fibrosarcoma homolog NM_005360Blood AR NCOR2 Nuclear receptor co-repressor 2 NM_006312 Blood CR ZFP106Zinc finger protein 106 homolog NM_022473 Blood CR RPL23 Ribosomalprotein L23 NM_000978 Blood CR CPVL Carboxypeptidase, vitellogenic-likeNM_019029 Blood CR ENO2 Enolase 2 (gamma, neuronal) NM_001975 Blood CRCAPN2 Calpain 2, (m/II) large subunit NM_001748 Blood CR FGFR4Fibroblast growth factor receptor 4 NM_002011 Blood CR CD68 CD68 antigenNM_001251 Blood CR HK3 Hexokinase 3 (white cell) NM_002115 Blood CRDUSP6 Dual specificity phosphatase 6 NM_001946 Blood CR IL6STInterleukin 6 signal transducer NM_002184 Blood CR LATS2 LATS, largetumor suppressor 2 NM_014572 Blood CR MIC2 CD99 antigen NM_002414 BloodCR MMP23B Matrix metalloproteinase 23B NM_006983 Blood CR ZNF511 Zincfinger protein 511 NM_145806 Blood CR ANXA5 Annexin A5 NM_001154 BloodCR ID2 Inhibitor of DNA binding 2 NM_002166 Blood CR PRKRIR RNAdependent p58 repressor NM_004705 Blood CR SGK Serum/glucocorticoidregulated kinase NM_005627 Blood CR S100A10 S100 calcium binding proteinA10 NM_002966 Blood CR CYP51 Cytochrome P450, family 51A NM_000786 BloodCR ITGA4 Integrin, alpha 4 (antigen CD49D) NM_000885 Blood CR ADAM10 Adisintegrin and metalloproteinase10 NM_001110 Blood CR HNRPK Nuclearribonucleoprotein K NM_031262 Blood CR ITGAV Integrin, alpha V (CD51)NM_002210 Blood CR JUN V-jun sarcoma virus 17 homolog NM_002228 Blood CRPRKAR2B Protein kinase regulator NM_002736 Blood CR TIE Tyrosine kinasewith Ig and EGF domains NM_005424 Blood CR IQGAP2 GTPase activatingprotein 2 NM_006633 Blood CR MAP4K1 Mitogen-activated protein kinase 1NM_007181 Blood CR ILF3 Interleukin enhancer binding factor 3 NM_012218Blood CR SGKL Serum/glucocorticoid regulated kinase-like NM_013257 BloodCR GLS Glutaminase NM_014905 Blood CR DPYD Dihydropyrimidinedehydrogenase NM_000110 Blood CR ACADM Acyl-Coenzyme A dehydrogenaseNM_000016 Biopsy DT AUTS2 Autism susceptibility candidate 2 NM_015570Biopsy DT CA2 Carbonic anhydrase II NM_000067 Biopsy DT CTNNA1 Catenin(cadherin-associated protein) NM_001903 Biopsy DT CXCL12 Stromalcell-derived factor 1 NM_000609 Biopsy DT DDR1 Discoidin domain receptorfamily, member 1 NM_013994 Biopsy DT DECR1 2,4-dienoyl CoA reductase 1,mitochondrial NM_001359 Biopsy DT DEDD Death effector domain containingNM_032998 Biopsy DT DPP4 Dipeptidylpeptidase 4 (CD26) NM_001935 BiopsyDT ITM2B Integral membrane protein 2B NM_021999 Biopsy DT KIAA0436L-type neutral amino acid transporter AB007896 Biopsy DT LDHB Lactatedehydrogenase B NM_002300 Biopsy DT LEPR Leptin receptor NM_002303Biopsy DT LRBA LPS-responsive vesicle trafficking NM_006726 Biopsy DTMUT Methylmalonyl Coenzyme A mutase NM_000255 Biopsy DT NAT1N-acetyltransferase 1 NM_000662 Biopsy DT NAT2 N-acetyltransferase 2NM_000015 Biopsy DT NUP50 Nucleoporin 50 kDa NM_153645 Biopsy DTPAFAH1B1 Platelet-activating factor NM_000430 Biopsy DT PDZK3 PDZ domaincontaining 3 NM_178140 Biopsy DT PLCL2 Phospholipase C-like 2 NM_015184Biopsy DT PPP2CB Protein phosphatase 2 NM_004156 Biopsy DT PRKCM Proteinkinase C, mu NM_002742 Biopsy DT PTPN3 Protein tyrosine phosphataseNM_002829 Biopsy DT REST RE1-silencing transcription factor NM_005612Biopsy DT SGCB Sarcoglycan, beta NM_000232 Biopsy DT SHB Src homology 2domain containing NM_003028 Biopsy DT SORL1 Sortilin-related receptor, LNM_003105 Biopsy DT SULT1E1 Sulfotransferase family 1E NM_005420 BiopsyDT CBL Cas-Br-Transforming sequence NM_005188 Biopsy DT CXCL1 Chemokine(C—X—C motif) ligand 1 NM_001511 Biopsy DT FGF2 Fibroblast growth factor2 (basic) NM_002006 Biopsy DT GPRK5 G protein-coupled receptor kinase 5NM_005308 Biopsy DT ITSN2 Intersectin 2 NM_006277 Biopsy DT BCL2L13BCL2-like 13 (apoptosis facilitator) AA279535 Biopsy BMI BDKRB2Bradykinin receptor B2 NM_000623 Biopsy BMI DDX3 DEAD/H(Asp-Glu-Ala-Asp/His) box 3 NM_001356 Biopsy BMI FOXM1 Forkhead box M1NM_021953 Biopsy BMI HMOX2 Heme oxygenase (decycling) 2 NM_002134 BiopsyBMI IFNGR1 Interferon gamma receptor 1 NM_000416 Biopsy BMI IGFBP1Insulin-like growth factor binding protein 1 NM_000596 Biopsy BMI IGFBP5Insulin-like growth factor binding protein 5 NM_000599 Biopsy BMI LRP2Low density lipoprotein-related protein 2 NM_004525 Biopsy BMI MCM7Minichromosome maintenance deficient 7 NM_182776 Biopsy BMI NPPBNatriuretic peptide precursor B NM_002521 Biopsy BMI NPR1 Natriureticpeptide receptor A NM_000906 Biopsy BMI PAXIP1L PAX transcriptionactivation interacting NM_007349 Biopsy BMI PDCD5 Programmed cell death5 NM_004708 Biopsy BMI RBX1 Ring-box 1 NM_014248 Biopsy BMI RPL27Ribosomal protein L27 NM_000988 Biopsy BMI SBA2 WD repeat and SOCS boxcontaining protein AA043793 Biopsy BMI SERPINB6 Proteinase inhibitor,clade B (ovalbumin) NM_004568 Biopsy BMI SLC22A5 Solute carrier family22 NM_003060 Biopsy BMI SLC38A2 Solute carrier family 38, member 2NM_018976 Biopsy BMI SMT3H2 Suppressor of MIF NM_006937 Biopsy BMI TJP4Tight junction protein 4 (peripheral) NM_080604 Biopsy BMI TP53INP1 p53inducible nuclear protein 1 NM_033285 Biopsy BMI BHLHB2 Basichelix-loop-helix domain containing NM_003670 Biopsy BMI CSPG2Chondroitin sulfate proteoglycan 2 NM_004385 Biopsy BMI GPD1Glycerol-3-phosphate dehydrogenase 1 NM_005276 Biopsy BMI GTPBP4 GTPbinding 4; Chronic renal failure gene NM_012341 Biopsy BMI HIF1AHypoxia-inducible factor 1, alpha NM_001530 Biopsy BMI MMP7 Matrixmetalloproteinase 7 NM_002423 Biopsy BMI SLC2A3 Facilitated glucosetransporter NM_006931 Biopsy BMI THBS1 Thrombospondin 1 NM_003246 BiopsyBMI TNC Tenascin C (hexabrachion) NM_002160 Biopsy BMI HLA-G HLA-Ghistocompatibility antigen, class I, G NM_002127 Blood TOL IGHG3 Igheavy constant gamma 3 AK097306 Blood TOL BUR1 Budding uninhibited (cellcycle regulator) NM_004336 Blood TOL CCNB2 Cyclin B2 NM_004701 Blood TOLTACSTD1 Tumor-associated calcium signaling NM_002354 Blood TOL DHRS2Dehydrogenase/reductase (SDR family) AK092834 Blood TOL BMP7 Bonemorphogenetic protein 7 NM_001719 Blood TOL AKR1C1 Aldo-keto reductasefamily 1C1 NM_001353 Blood TOL B4GALT2 UDP-Gal 1,4-galactosyltransferaseNM_003780 Blood TOL TCEB3 Transcription elongation factor B (SIII)NM_003198 Blood TOL MLPH Melanophilin NM_024101 Blood TOL SERPINH2 Heatshock protein 47 (proteinase inhibitor) NM_001235 Blood TOL RRM2Ribonucleotide reductase M2 polypeptide NM_001034 Blood TOL SERPINA3Alpha-1 antiproteinase, antitrypsin NM_001085 Blood TOL SERPINA5 Alpha-1antiproteinase, antitrypsin NM_000624 Blood TOL CTNNAL1 Catenin(cadherin-associated protein) NM_003798 Blood TOL SPARC Secretedprotein, cysteine-rich (osteonectin) NM_003118 Blood TOL C1S C1Scomplement component 1 NM_001734 Blood TOL SRPUL SRPUL sushi-repeatprotein NM_006307 Blood TOL MMP2 Matrix metalloproteinase 2 NM_004530Blood TOL SLC7A7 Cationic amino acid transporter NM_003982 Blood TOLEPOR Erythropoietin receptor NM_000121 Blood TOL PRAME Preferentiallyexpressed antigen in melanoma NM_006115 Blood TOL AFP Alpha-fetoproteinNM_001134 Blood TOL MAPK9 Mitogen-activated protein kinase 9 NM_002752Blood TOL NR2F2 Nuclear receptor subfamily 2F2 NM_021005 Blood TOL PFN2Profilin 2 NM_053024 Blood TOL SLC38A6 Solute carrier family 38, member6 BC050349 Blood TOL MYOM2 Myomesin (M-protein) 2, 165 kDa NM_003970Blood TOL RBP1 Retinol binding protein 1, cellular NM_002899 Blood TOLTK1 Thymidine kinase 1, soluble NM_003258 Blood TOL IFITM3 Interferoninduced transmembrane protein 3 NM_021034 Blood TOL APOH ApolipoproteinH (beta-2-glycoprotein I) NM_000042 Blood TOL EVI2A Ecotropic viralintegration site 2A NM_014210 Blood TOL CD9 CD9 antigen (p24) NM_001769Blood TOL NKG7 Natural killer cell group 7 sequence NM_005601 Blood TOLCDKN3 Cyclin-dependent kinase inhibitor 3 NM_005192 Blood TOL TCL1AT-cell leukemia/lymphoma 1A NM_021966 Blood TOL PYCR1Pyrroline-5-carboxylate reductase 1 NM_153824 Blood TOL TM4SF5Transmembrane 4 superfamily member 5 NM_003963 Blood TOL GAGEB1 Gantigen, family B, 1 (prostate associated) NM_003785 Blood TOL PCP4Purkinje cell protein 4 NM_006198 Blood TOL LGMN Legumain NM_005606Blood TOL PIR Pirin (iron-binding nuclear protein) NM_178238 Blood TOLPAICS Phosphoribosylaminoimidazole carboxylase NM_006452 Blood TOLIGFBP3 Insulin-like growth factor binding protein 3 NM_000598 Blood TOLPSMB9 Proteasome subunit NM_002800 Blood TOL N33 Putative prostatecancer tumor suppressor NM_006765 Blood TOL DP1 Polyposis locus protein1 (DP1) NM_005669 Blood TOL TFDP1 Transcription factor Dp-1 NM_007111Blood TOL OSF-2 OSF-2 osteoblast specific factor 2 NM_000358 Blood TOLCOL3A1 Collagen, type III, alpha 1 NM_000090 Blood TOL TIMP3 TIMP3tissue inhibitor of metalloproteinase 3 NM_000362 Blood TOL SPP1Osteopontin, early T-lymphocyte activation 1 NM_000582 Blood TOL NQO1NQO1 NAD(P)H dehydrogenase NM_000903 Blood TOL TOP2A Topoisomerase (DNA)II alpha 170 kDa NM_001067 Blood TOL CCND2 Cyclin D2 NM_001759 Blood TOLCNN3 CNN3 calponin 3, acidic AI969128 NM_001839 Blood TOL COL6A1Collagen, type VI, alpha 1 NM_001848 Blood TOL CTGF Connective tissuegrowth factor NM_001901 Blood TOL EGR1 Early growth response 1 (EGR1)NM_001964 Blood TOL SDC2 Syndecan 2 NM_002998 Blood TOL TCF3Transcription factor 3 NM_003200 Blood TOL TFAP2C Transcription factorAP-2 gamma NM_003222 Blood TOL NRP1 Neuropilin 1 NM_003873 Blood TOLGITR TNF receptor superfamily18 (TNFRSF18) NM_004195 Blood TOL COL6A3Collagen, type VI, alpha 3 NM_004369 Blood TOL EPHA2 EPHA2 EphA2NM_004431 Blood TOL PDE1A ARHE ras homolog gene family NM_005168 BloodTOL FAT Tumor suppressor homolog 1 NM_005245 Blood TOL KIFC3 Kinesinfamily member C3 NM_005550 Blood TOL NR2F1 Nuclear receptor subfamily2F1 NM_005654 Blood TOL CAP2 CAP, adenylate cyclase-associated 2NM_006366 Blood TOL BACE2 Beta-site APP-cleaving enzyme 2 NM_012105Blood TOL FADS1 Fatty acid desaturase 1 NM_013402 Blood TOL MELKMaternal embryonic leucine zipper kinase NM_014791 Blood TOL DKK3Dickkopf homolog 3 (Xenopus laevis) NM_015881 Blood TOL CCNB1 Cyclin B1NM_031966 Blood TOL CALD1 Caldesmon 1 NM_033138 Blood TOL CASP1 Caspase1, (interleukin 1b convertase) NM_033292 Blood TOL KNSL5 Kinesin-like 5(mitotic kinesin-like protein 1) NM_138555 Blood TOL STK6Serine/threonine kinase 6 NM_198433 Blood TOL CD59 CD59 antigen p18-20NM_203330 Blood TOL FN1 Fibronectin 1 NM_212482 Blood TOL SERPINE2Serine proteinase inhibitor NM_006216 Blood TOL CDH2 Cadherin 2, type 1,N-cadherin NM_001792 Blood TOL CCNE1 Cyclin E1 NM_001238 Blood TOLSEMA3F Ig short basic domain, secreted NM_004186 Blood TOL MAD2L1 MAD2mitotic arrest deficient-like 1 NM_002358 Blood TOL CYR61 Cysteine-rich,angiogenic inducer, 61 NM_001554 Blood TOL TNFRSF7 CD27 TNF receptorsuperfamily 7 NM_001242 Blood TOL FOXP3 Forkhead box P3 (FOXP3), mRNANM_014009 Blood TOL ABCA4 ATP-binding cassette, sub-family A (ABC1)NM_000350 Biopsy Control HNK-1 HNK-1 sulfotransferase AF033827 BiopsyControl UCP2 Uncoupling protein 2 NM_003355 Biopsy Control DAB2Mitogen-responsive phosphoprotein NM_001343 Biopsy Control AQP3Aquaporin 3 NM_004925 Biopsy Control CRABP1 Cellular retinoic acidbinding protein 1 NM_004378 Biopsy Control KCNAB2 Potassiumvoltage-gated channel NM_003636 Biopsy Control TNNT2 Troponin T2,cardiac NM_000364 Biopsy Control APP Amyloid beta (A4) precursor proteinNM_000484 Biopsy Control FABP3 Fatty acid binding protein 3 NM_004102Biopsy Control PODXL Podocalyxin-like NM_005397 Biopsy Control ALPIAlkaline phosphatase, intestinal NM_001631 Biopsy Control MAPTMicrotubule-associated protein tau NM_005910 Biopsy Control KHKKetohexokinase (fructokinase) NM_000221 Biopsy Control 18S 18s ribosomalRNA M10098 All Control ACTB Actin, beta NM_001101 All Control GAPDGlyceraldehyde-3-phosphate dehydrogenase NM_002046 All Control GSUSBGlucuronidase, beta NM_000181 All Control HPRT1 Hypoxanthinephosphoribosyltransferase 1 NM_000194 All Control SCYA3 Chemokine (C—Cmotif) ligand 3 NM_002983 All Control LMO2 LIM domain only 2 (LMO2)NM_005574 All Control BCL6 B-cell CLL/lymphoma 6 NM_001706 All ControlIkB2 NFkB enhancer in B-cells inhibitor NM_020529 All Control APCAdenomatosis polyposis coli NM_000038 All Control BAG2 BCL2-associatedathanogene 2 (BAG2) NM_004282 All Control CREBBP CREB binding proteinNM_004380 All Control KLRB1 Killer cell lectin-like receptor B1NM_002258 All Control TRADD TNFRSF1A-associated via death domainNM_003789 All Control CXCL14 Chemokine (C—X—C motif) ligand 14 NM_004887All Control IL1A Interleukin 1, alpha NM_000575 All Control MMP1 Matrixmetalloproteinase 1 NM_002421 All Control MMP9 Matrix metalloproteinase9 NM_004994 All Control VEGFC Vascular endothelial growth factor CNM_005429 All Control CD8A CD8 antigen, alpha polypeptide (p32)NM_171827 Blood Control CD3G CD3G antigen, gamma (TiT3 complex)NM_000073 Blood Control CD44 CD44 antigen NM_000610 Blood Control CD4CD4 antigen (p55) NM_000616 Blood Control CD3D CD3D antigen, delta (TiT3complex) NM_000732 Blood Control CD3E CD3E antigen, epsilon (TiT3complex) NM_000733 Blood Control CD3Z CD3Z antigen, zeta (TiT3 complex)NM_000734 Blood Control CD19 CD19 antigen NM_001770 Blood Control B220Protein tyrosine phosphatase receptor NM_002838 Blood Control CD138CD138 syndecan 1 (SDC1) NM_002997 Blood Control CD43 Sialophorin (CD43)NM_003123 Blood Control CD8B1 CD8 antigen, beta polypeptide 1 (p37)NM_004931 Blood Control API5 Apoptosis inhibitor 5 NM_006595 All Lit.Axin1 Axin 1 NM_003502 All Lit. Axin2 Axin 2 (conductin, axil) NM_004655All Lit. BAD BCL2-antagonist of cell death NM_032989 All Lit. BIKBCL2-interacting killer (apoptosis-inducing) NM_001197 All Lit. BMP4Bone morphogenetic protein 4 NM_001202 All Lit. BTG1 B-celltranslocation gene 1 NM_001731 All Lit. CASP10 Caspase 10,apoptosis-related cysteine protease NM_001230 All Lit. CASP3 Caspase 3,apoptosis-related cysteine protease NM_004346 All Lit. CASP4 Caspase 4,apoptosis-related cysteine protease NM_001225 All Lit. CASP7 Caspase 7,apoptosis-related cysteine protease NM_001227 All Lit. CASP9 Caspase 9,apoptosis-related cysteine protease NM_001229 All Lit. CCL18 Chemokine(C—C motif) ligand 18 NM_002988 All Lit. CD161 Killer cell lectin-likereceptor B1 BC027885 All Lit. CD20 Membrane-spanning 4A1 NM_152866 AllLit. CD22 CD22 antigen NM_001771 All Lit. CD48 CD48 antigen (B-cellmembrane protein) NM_001778 All Lit. CD80 CD80 antigen (B7-1 antigen)NM_005191 All Lit. CDA08 T-cell immunomodulatory protein NM_030790 AllLit. CDC2 Cell division cycle 2, G1 to S and G2 to M NM_001786 All Lit.CDw108 Semaphorin Ig and GPI membrane anchor 7A, NM_003612 All Lit.CDW52 CDW52 antigen (CAMPATH-1 antigen) NM_001803 All Lit. CIS4 STATinduced STAT inhibitor-4 NM_004232 All Lit. CTLA4 CytotoxicT-lymphocyte-associated protein 4 NM_005214 All Lit. DAD1 Defenderagainst cell death 1 NM_001344 All Lit. DAP3 Death associated protein 3NM_033657 All Lit. DAPK2 Death-associated protein kinase 2 NM_014326 AllLit. DAPK3 Death-associated protein kinase 3 NM_001348 All Lit. DAXXDeath-associated protein 6 NM_001350 All Lit. EBF Early B-cell factorNM_024007 All Lit. FCGR1A Fc fragment of IgG (receptor for CD64)NM_000566 All Lit. GADD45B Growth arrest and DNA-damage-inducibleNM_015675 All Lit. GSR Glutathione reductase NM_000637 All Lit. GZMAGranzyme A NM_006144 All Lit. GZMB Granzyme B NM_004131 All Lit. GzmcGranzyme C M18459 All Lit. GZMK Granzyme K NM_002104 All Lit. HLA-E MHCclass I, E NM_005516 All Lit. ICAM1 Intercellular adhesion molecule 1(CD54) NM_000201 All Lit. ICAM3 Intercellular adhesion molecule 3NM_002162 All Lit. IFI16 Interferon, gamma-inducible protein 16NM_005531 All Lit. IFI35 Interferon-induced protein 35 NM_005533 AllLit. IFNG Interferon, gamma NM_000619 All Lit. IGBP1 Ig (CD79A) bindingprotein 1 NM_001551 All Lit. IGJ Ig J polypeptide, linker proteinNM_144646 All Lit. IK IK cytokine, down-regulator of HLA II NM_006083All Lit. IL2RA Interleukin 2 receptor, alpha NM_000417 All Lit. IL4RInterleukin 4 receptor NM_000418 All Lit. IL6 Interleukin 6 (interferon,beta 2) NM_000600 All Lit. IL7R Interleukin 7 receptor NM_002185 AllLit. IL8RB Interleukin 8 receptor, beta NM_001557 All Lit. IRF1Interferon regulatory factor 1 NM_002198 All Lit. ITGAE Integrin, alphaE (CD103) NM_002208 All Lit. JAK1 Janus kinase 1 NM_002227 All Lit. JAK2Janus kinase 2 NM_004972 All Lit. MADH2 SMAD, mothers against DPPNM_005901 All Lit. MAPK3 Mitogen-activated protein kinase 3 NM_002746All Lit. MDM2 p53 binding protein NM_002392 All Lit. MHC2TA MHC class IItransactivator NM_000246 All Lit. NK4 Natural killer cell transcript 4NM_004221 All Lit. NMI N-myc (and STAT) interactor NM_004688 All Lit.PCNA Proliferating cell nuclear antigen NM_002592 All Lit. PDCD2Programmed cell death 2 NM_002598 All Lit. PDCD7 Programmed cell death 7NM_005707 All Lit. PDCD8 Programmed cell death 8 NM_004208 All Lit.PDGFRB Platelet-derived growth factor receptor NM_002609 All Lit. RhoARas homolog gene family, member A NM_001664 All Lit. SIMRP7 Multidrugresistance-associated protein 7 NM_033450 All Lit. SOD2 Superoxidedismutase 2, mitochondrial NM_000636 All Lit. SSI-1 suppressor ofcytokine signaling 1 NM_003745 All Lit. STAT2 Signal transducer2, 113kDa NM_005419 All Lit. STAT3 Signal transducer 3 (acute-phase responsefactor) NM_139276 All Lit. STAT4 Signal transducer 4 NM_003151 All Lit.STAT5A Signal transducer 5A NM_003152 All Lit. STAT5B Signal transducera5B NM_012448 All Lit. STK21 Rho-interacting NM_007174 All Lit. TA-LRRPTNF receptor-associated factor 6 NM_145803 All Lit. TCRA T-cell receptoractive alpha-chain M12423 All Lit. TCRB T cell receptor beta locusX60096 All Lit. TCRD T-cell receptor delta chain (VJC-region) M21624 AllLit. TCRG T cell receptor gamma locus X06774 All Lit. TFRC Transferrinreceptor (p90, CD71) NM_003234 All Lit. TGFA Transforming growth factor,alpha NM_003236 All Lit. TGFB2 Transforming growth factor, beta 2NM_003238 All Lit. THBS2 Thrombospondin 2 NM_003247 All Lit. TIA1Cytotoxic granule-associated RNA binding NM_022173 All Lit. TIEG2 TGFBinducible early growth response 2 NM_003597 All Lit. TLR5 Toll-likereceptor 5 NM_003268 All Lit. TNFRSF1A TNF receptor superfamily, member1A NM_001065 All Lit. TNFRSF1B TNF receptor superfamily, member 1BNM_001066 All Lit. TNFSF7 TNF (ligand) superfamily, member 7 NM_001252All Lit. TP53BP1 Tumor protein p53 binding protein, 1 NM_005657 All Lit.TP53BP2 Tumor protein p53 binding protein, 2 NM_005426 All Lit. TRAF1TNF receptor-associated factor 1 NM_005658 All Lit. TRAF2 TNFreceptor-associated factor 2 NM_021138 All Lit. TRAF3 TNFreceptor-associated factor 3 NM_003300 All Lit. TRAF4 TNFreceptor-associated factor 4 NM_004295 All Lit. TRAP1 TNFreceptor-associated protein 1 NM_004257 All Lit. TTK TTK protein kinaseNM_003318 All Lit. UBE1L Ubiquitin-activating enzyme E1-like NM_003335All Lit. VPREB3 Pre-B lymphocyte gene 3 NM_013378 All Lit. WNT1 MMTVintegration site (WNT1) NM_005430 All Lit. ACE1 Ig receptor (PIGR) IgAnephritis NM_002644 All Lit. BAX BCL2-associated X protein NM_138763 AllLit. BCL2 B-cell CLL/lymphoma 2 NM_000633 All Lit. C3 Complementcomponent 3 NM_000064 All Lit. CD28 CD28 antigen (Tp44) NM_006139 AllLit. CD86 CD86 antigen (B7-2 antigen) NM_006889 All Lit. ICOS InducibleT-cell co-stimulator NM_012092 All Lit. IL10 Interleukin 10 NM_000572All Lit. IL15 Interleukin 15 NM_000585 All Lit. IL2 Interleukin 2NM_000586 All Lit. IL4 Interleukin 4 NM_000589 All Lit. IL7 Interleukin7 NM_000880 All Lit. IL8 Interleukin 8 NM_000584 All Lit. PRF1 Perforin1 (pore forming protein) NM_005041 All Lit. RANTES Chemokine (C—C motif)ligand 5 (CCL5) NM_002985 All Lit. TBET Th1-specific T-box transcriptionfactor NM_013351 All Lit. TGFB1 TGF beta 1 NM_000660 All Lit. TNF TNFsuperfamily, member 2 NM_000594 All Lit. TNFB Lymphotoxin alpha (TNF1 orLTA) NM_000595 All Lit. TNFRSF5 CD40 TNF receptor superfamily 5NM_001250 All Lit. TNFRSF6 CD95 = Fas TNF receptor superfamily 6NM_000043 All Lit. VEGF Vascular endothelial growth factor NM_003376 AllLit.

In certain embodiments, a collection of genes from Table 3 is assayed,where in these embodiments the number of genes from Table 3 may be atleast about 5%, at least about 10%, at least about 25%, at least about50%, at least about 75%, at least about 90% or more, including all ofthe genes from Table 3.

In certain embodiments, the expression profile obtained is a genomic ornucleic acid expression profile, where the amount or level of one ormore nucleic acids in the sample is determined, e.g., the nucleic acidtranscript of the gene of interest. In these embodiments, the samplethat is assayed to generate the expression profile employed in thediagnostic methods is one that is a nucleic acid sample. The nucleicacid sample includes a plurality or population of distinct nucleic acidsthat includes the expression information of the phenotype determinativegenes of interest of the cell or tissue being diagnosed. The nucleicacid may include RNA or DNA nucleic acids, e.g., mRNA, cRNA, cDNA etc.,so long as the sample retains the expression information of the hostcell or tissue from which it is obtained. The sample may be prepared ina number of different ways, as is known in the art, e.g., by mRNAisolation from a cell, where the isolated mRNA is used as is, amplified,employed to prepare cDNA, cRNA, etc., as is known in the differentialexpression art. In certain embodiments, the sample is prepared from acell or tissue harvested from a subject to be diagnosed, e.g., viabiopsy of tissue, using standard protocols, where cell types or tissuesfrom which such nucleic acids may be generated include any tissue inwhich the expression pattern of the to be determined phenotype exists,including, but not limited to, peripheral blood lymphocyte cells, etc,as reviewed above.

The expression profile may be generated from the initial nucleic acidsample using any convenient protocol. While a variety of differentmanners of generating expression profiles are known, such as thoseemployed in the field of differential gene expression analysis, onerepresentative and convenient type of protocol for generating expressionprofiles is array-based gene expression profile generation protocols. Incertain embodiments, such applications are hybridization assays in whicha nucleic acid array that displays “probe” nucleic acids for each of thegenes to be assayed/profiled in the profile to be generated is employed.In these assays, a sample of target nucleic acids is first prepared fromthe initial nucleic acid sample being assayed, where preparation mayinclude labeling of the target nucleic acids with a label, e.g., amember of signal producing system. Following target nucleic acid samplepreparation, the sample is contacted with the array under hybridizationconditions, whereby complexes are formed between target nucleic acidsthat are complementary to probe sequences attached to the array surface.The presence of hybridized complexes is then detected, eitherqualitatively or quantitatively. Specific hybridization technology whichmay be practiced to generate the expression profiles employed in thesubject methods includes the technology described in U.S. Pat. Nos.5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;5,800,992; the disclosures of which are herein incorporated byreference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO97/27317; EP 373 203; and EP 785 280. In these methods, an array of“probe” nucleic acids that includes a probe for each of the phenotypedeterminative genes whose expression is being assayed is contacted withtarget nucleic acids as described above. Contact is carried out underhybridization conditions, e.g., stringent hybridization conditions, andunbound nucleic acid is then removed.

The resultant pattern of hybridized nucleic acid provides informationregarding expression for each of the genes that have been probed, wherethe expression information is in terms of whether or not the gene isexpressed and, typically, at what level, where the expression data,i.e., expression profile (e.g., in the form of a transcriptosome), maybe both qualitative and quantitative.

Alternatively, non-array based methods for quantitating the levels ofone or more nucleic acids in a sample may be employed, includingquantitative PCR, and the like.

Where the expression profile is a protein expression profile, anyconvenient protein quantitation protocol may be employed, where thelevels of one or more proteins in the assayed sample are determined.Representative methods include, but are not limited to: proteomicarrays, flow cytometry, standard immunoassays (e.g., ELISA assays),protein activity assays, including multiplex protein activity assays,etc.

Following obtainment of the expression profile from the sample beingassayed, the expression profile is compared with a reference or controlprofile to determine the particular graft tolerant/intolerant phenotypeof the cell or tissue, and therefore host, from which the sample wasobtained/derived. The terms “reference” and “control” as used hereinmean a standardized pattern of gene expression or levels of expressionof certain genes to be used to interpret the expression signature of agiven patient and assign a graft tolerant/intolerant phenotype thereto.The reference or control profile may be a profile that is obtained froma cell/tissue known to have the desired phenotype, e.g., tolerantphenotype, and therefore may be a positive reference or control profile.In addition, the reference/control profile may be from a cell/tissueknown to not have the desired phenotype, e.g., an intolerant phenotype,and therefore be a negative reference/control profile.

In certain embodiments, the obtained expression profile is compared to asingle reference/control profile to obtain information regarding thephenotype of the cell/tissue being assayed. In yet other embodiments,the obtained expression profile is compared to two or more differentreference/control profiles to obtain more in depth information regardingthe phenotype of the assayed cell/tissue. For example, the obtainedexpression profile may be compared to a positive and negative referenceprofile to obtain confirmed information regarding whether thecell/tissue has the phenotype of interest.

The comparison of the obtained expression profile and the one or morereference/control profiles may be performed using any convenientmethodology, where a variety of methodologies are known to those ofskill in the array art, e.g., by comparing digital images of theexpression profiles, by comparing databases of expression data, etc.Patents describing ways of comparing expression profiles include, butare not limited to, U.S. Pat. Nos. 6,308,170 and 6,228,575, thedisclosures of which are herein incorporated by reference. Methods ofcomparing expression profiles are also described above.

The comparison step results in information regarding how similar ordissimilar the obtained expression profile is to the control/referenceprofile(s), which similarity/dissimilarity information is employed todetermine the phenotype of the cell/tissue being assayed and therebyevaluate graft survival in the subject. For example, similarity with apositive control indicates that the assayed cell/tissue has a graftsurvival phenotype. Likewise, similarity with a negative controlindicates that the assayed cell/tissue has a graft loss phenotype.

Depending on the type and nature of the reference/control profile(s) towhich the obtained expression profile is compared, the above comparisonstep yields a variety of different types of information regarding thecell/tissue that is assayed. As such, the above comparison step canyield a positive/negative determination of a graft survival phenotype ofan assayed cell/tissue. In many embodiments, the above-obtainedinformation about the cell/tissue being assayed is employed to diagnosea host, subject or patient with respect to graft survival, as describedabove. In certain embodiments, the determination/prediction of graftsurvival and loss can be coupled with a determination of additionalcharacteristics of the graft and function thereof. For example, incertain embodiments one can predict not only whether graft loss willoccur, but the mechanism of graft loss, e.g., via CAN or DT. The first 9genes in the cluster illustrated in FIG. 4 are highly-differentiallyexpressed between CAN and DT. As such, evaluating one or more of thesegenes permits these two overlapping conditions to be readilydistinguished, such that one can readily determine the presence of CANor DT.

The subject methods further find use in pharmacogenomic applications. Inthese applications, a subject/host/patient is first diagnosed for graftfunction according to the subject invention, and then treated using aprotocol determined, at least in part, on the results of the diagnosis.For example, a host may be evaluated for the presence of absence of thegraft survival phenotype using a protocol such as the diagnosticprotocol described in the preceding section. The subject may then betreated using a protocol whose suitability is determined using theresults of the diagnosis step. In embodiments, where the host isevaluated for the presence or absence of CAN or DT, treatment protocolsmay correspondingly be adjusted based on the obtained results. Forexample, where the subject methods are employed to determine thepresence of CAN, immunosuppressive therapy can be modulated, e.g.,increased or drugs changed, as is known in the art for the treatment ofCAN. Likewise, where the subject methods are employed and detect thepresence of DT, the immunosuppressive therapy can be reduced in order totreat the DT. In practicing the subject methods, a subject is typicallyscreened for the presence of a graft survival or loss phenotypefollowing receipt of a graft or transplant. The subject may be screenedonce or serially following transplant receipt, e.g., weekly, monthly,bimonthly, half-yearly, yearly, etc. In certain embodiments, the subjectis screened following occurrence of acute rejection (AR). In suchembodiments, the methods are employed to evaluate, e.g., predict,ultimate graft loss or survival in the subject following AR.

The subject methods may be employed with a variety of different types oftransplant subjects. In many embodiments, the subjects are within theclass mammalian, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), lagomorpha (e.g. rabbits)and primates (e.g., humans, chimpanzees, and monkeys). In certainembodiments, the animals or hosts, i.e., subjects (also referred toherein as patients) will be humans.

The methods may be used to evaluate survival of a variety of differenttypes of grafts. Grafts of interest include, but are not limited to:transplanted heart, kidney, lung, liver, pancreas, pancreatic islets,brain tissue, stomach, large intestine, small intestine, cornea, skin,trachea, bone, bone marrow, muscle, bladder or parts thereof.

Databases of Expression Profiles of Phenotype Determinative Genes

Also provided are databases of expression profiles of graft survivaland/or graft loss phenotype determinative genes. Such databases willtypically comprise expression profiles of various cells/tissues havinggraft tolerant phenotypes, negative expression profiles, etc., wheresuch profiles are further described below.

The expression profiles and databases thereof may be provided in avariety of media to facilitate their use. “Media” refers to amanufacture that contains the expression profile information of thepresent invention. The databases of the present invention can berecorded on computer readable media, e.g. any medium that can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. One of skill in theart can readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising arecording of the present database information. “Recorded” refers to aprocess for storing information on computer readable medium, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g. word processing text file, database format, etc.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the informationof the present invention. The minimum hardware of the computer-basedsystems of the present invention comprises a central processing unit(CPU), input means, output means, and data storage means. A skilledartisan can readily appreciate that any one of the currently availablecomputer-based system are suitable for use in the present invention. Thedata storage means may comprise any manufacture comprising a recordingof the present information as described above, or a memory access meansthat can access such a manufacture.

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. One format for an output means ranksexpression profiles possessing varying degrees of similarity to areference expression profile. Such presentation provides a skilledartisan with a ranking of similarities and identifies the degree ofsimilarity contained in the test expression profile.

Reagents, Systems and Kits

Also provided are reagents, systems and kits thereof for practicing oneor more of the above-described methods. The subject reagents, systemsand kits thereof may vary greatly. Reagents of interest include reagentsspecifically designed for use in production of the above-describedexpression profiles of phenotype determinative genes, i.e., a geneexpression evaluation element made up of one or more reagents. The termsystem refers to a collection of reagents, however compiled, e.g., bypurchasing the collection of reagents from the same or differentsources. The term kit refers to a collection of reagents provided, e.g.,sold, together.

One type of such reagent is an array of probe nucleic acids in which thephenotype determinative genes of interest are represented. A variety ofdifferent array formats are known in the art, with a wide variety ofdifferent probe structures, substrate compositions and attachmenttechnologies. Representative array structures of interest include thosedescribed in U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049;5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839;5,580,732; 5,661,028; 5,800,992; the disclosures of which are hereinincorporated by reference; as well as WO 95/21265; WO 96/31622; WO97/10365; WO 97/27317; EP 373 203; and EP 785 280.

In certain embodiments, the arrays include probes for at least 1 of thegenes listed in Tables 1 and/or 2. In certain embodiments, the number ofgenes that are from Tables 1 and/or 2 that is represented on the arrayis at least 5, at least 10, at least 25, at least 50, at least 75 ormore, including all of the genes listed in Tables 1 and/or 2. Thesubject arrays may include only those genes that are listed in Tables 1and/or 2, or they may include additional genes that are not listed inTables 1 and/or 2, such as probes for genes whose expression pattern canbe used to evaluate additional transplant characteristics, including butnot limited to: acute rejection; chronic allograft injury (chronicrejection) in blood; immunosuppressive drug toxicity or adverse sideeffects including drug-induced hypertension; age or body mass indexassociated genes that correlate with renal pathology or account fordifferences in recipient age-related graft acceptance; immune tolerancemarkers in whole blood; genes found in literature surveys with immunemodulatory roles that may play a role in transplant outcomes (see e.g.,Table 3 for a list of representative additional genes); as well as otherarray assay function related genes, e.g., for assessing sample quality(3′- to 5′-bias in probe location), sampling error in biopsy-basedstudies, cell surface markers, and normalizing genes for calibratinghybridization results; and the like. Where the subject arrays includeprobes for such additional genes, in certain embodiments the number % ofadditional genes that are represented and are not directly or indirectlyrelated to transplantation does not exceed about 50%, usually does notexceed about 25%. In certain embodiments where additional genes areincluded, a great majority of genes in the collection are transplantcharacterization genes, where by great majority is meant at least about75%, usually at least about 80% and sometimes at least about 85, 90, 95%or higher, including embodiments where 100% of the genes in thecollection are phenotype determinative genes. Transplantcharacterization genes are genes whose expression can be employed tocharacterize transplant function in some manner, e.g., presence ofrejection, etc.

Another type of reagent that is specifically tailored for generatingexpression profiles of phenotype determinative genes is a collection ofgene specific primers that is designed to selectively amplify suchgenes. Gene specific primers and methods for using the same aredescribed in U.S. Pat. No. 5,994,076, the disclosure of which is hereinincorporated by reference. Of particular interest are collections ofgene specific primers that have primers for at least 1 of the geneslisted in one Tables 1 and/or 2, often a plurality of these genes, e.g.,at least 2, 5, 10, 15 or more. In certain embodiments, the number ofgenes that are from Tables 1 and/or 2 that have primers in thecollection is at least 5, at least 10, at least 25, at least 50, atleast 75 or more, including all of the genes listed in Tables 1 and/or2. The subject gene specific primer collections may include only thosegenes that are listed in Tables 1 and/or 2, or they may include primersfor additional genes that are not listed in Tables 1 and/or 2, such asprobes for genes whose expression pattern can be used to evaluateadditional transplant characteristics, including but not limited to:acute rejection; chronic allograft injury (chronic rejection) in blood;immunosuppressive drug toxicity or adverse side effects includingdrug-induced hypertension; age or body mass index associated genes thatcorrelate with renal pathology or account for differences in recipientage-related graft acceptance; immune tolerance markers in whole blood;genes found in literature surveys with immune modulatory roles that mayplay a role in transplant outcomes (see e.g., Table 3 for a list ofrepresentative additional genes); as well as other array assay functionrelated genes, e.g., for assessing sample quality (3′- to 5′-bias inprobe location), sampling error in biopsy-based studies, cell surfacemarkers, and normalizing genes for calibrating hybridization results;and the like. Where the subject arrays include probes for suchadditional genes, in certain embodiments the number % of additionalgenes that are represented and are not directly or indirectly related totransplantation does not exceed about 50%, usually does not exceed about25%. In certain embodiments where additional genes are included, a greatmajority of genes in the collection are transplant characterizationgenes, where by great majority is meant at least about 75%, usually atleast about 80% and sometimes at least about 85, 90, 95% or higher,including embodiments where 100% of the genes in the collection arephenotype determinative genes.

The systems and kits of the subject invention may include theabove-described arrays and/or gene specific primer collections. Thesystems and kits may further include one or more additional reagentsemployed in the various methods, such as primers for generating targetnucleic acids, dNTPs and/or rNTPs, which may be either premixed orseparate, one or more uniquely labeled dNTPs and/or rNTPs, such asbiotinylated or Cy3 or Cy5 tagged dNTPs, gold or silver particles withdifferent scattering spectra, or other post synthesis labeling reagent,such as chemically active derivatives of fluorescent dyes, enzymes, suchas reverse transcriptases, DNA polymerases, RNA polymerases, and thelike, various buffer mediums, e.g. hybridization and washing buffers,prefabricated probe arrays, labeled probe purification reagents andcomponents, like spin columns, etc., signal generation and detectionreagents, e.g. streptavidin-alkaline phosphatase conjugate,chemifluorescent or chemiluminescent substrate, and the like.

The subject systems and kits may also include a phenotype determinationelement, which element is, in many embodiments, a reference or controlexpression profile that can be employed, e.g., by a suitable computingmeans, to make a phenotype determination based on an “input” expressionprofile, e.g., that has been determined with the above described geneexpression evaluation element. Representative phenotype determinationelements include databases of expression profiles, e.g., reference orcontrol profiles, as described above.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL I. Introduction

The objective of this study was to determine whether gene expressionmarkers could be identified in RNA extracted from peripheral bloodleukocytes (PBL) or renal biopsies predictive of future graft lossfollowing AR.

II. Array Experiments

Each microarray contained approximately 32,000 DNA spots representingapproximately 12,440 human genes. Total RNA was isolated (Tri Reagent;MRC Inc., Cincinnati, Ohio) from buffy coats isolated from whole bloodsamples. A common reference RNA pool (Perou et al., Nature (2000)406:747-52) was used as an internal standard. Sample or reference RNAwere subjected to two successive rounds of amplification beforehybridization to microarrays using an improved protocol based on themethod described by Wang et al (please provide entire cite). Array datafor 62 renal biopsy samples and 56 whole blood samples were stored inthe Stanford Microarray database (Sherlock et al., Nuc. Acids Res.(2001) 29:152-55) and gene lists filtered at retrieval to provideexpression markers with high fidelity. The two groups of samples wereanalyzed in two separate studies. All PBL were used for initialunsupervised hierarchical clustering (Eisen et al., Proc. Nat'l Acad.Sci. USA (1998) 95:14863-8), for subsequent supervised analyses betweengroups (Significance Analysis of Microarrays; SAM (Tusher et al., Proc.Nat'l Acad. Sci. USA (2001) 98:5116-21).

III. Customizing a Minimal Gene-Set for AR Class Prediction and RiskAssessment

We used Predictive Analysis of Microarrays (PAM) (Tusher et al., supra)to identify only 97 genes within the renal biopsy dataset, allhaving >5-fold difference in expression level, which classify ourlearning set of 26 AR samples with 100% concordance to assignedphenotype. Another analysis using a larger set of 3,170 differentiallyexpressed genes identifies the 33 classifiers with similar power (FIGS.1A and 1B). Reproducibility of the diagnostic signature, in particularwithin the majority of the AR-1 samples, is evident by the shortbranches in the cluster dendogram. AR expression overlaps with theinnate immune response to infection, as evidenced by cluster analysisand by differential expression of several TGF-β-modulated genesincluding RANTES, MIC-1, several cytokines, chemokines, andcell-adhesion molecules. AR-1 is the most severe class with the highestrate of graft loss and highest expression of B-cell specific genes. AR-2resembles a drug-toxicity signature and also co-clusters with patientswith active viral infections. The most striking feature of AR-3 is theexpression of genes involved in cellular proliferation and cell cyclingsuggesting active tissue repair and regeneration. The presence ofproliferating-cell nuclear antigen (PCNA), a marker of cellproliferation, was confirmed in all AR-3 samples tested (Sarwal et al.New Engl. J. Med. 2003 349(2):125-38).

The PAM classification scores grouped the samples with 100% concordanceto assigned classes and reported scores are aligned with the clusteredsamples (FIG. 1B). In addition, all 33 genes selected by PAM haveSignificance Analysis of Microarrays significance scores of 0.09% orlower suggesting that they would be highly significant biomarkers for acustomized array list.

A. PAM Class Prediction—

PAM class prediction has also proven to be a powerful approach toidentify putative biomarkers for graft recovery and graft loss. We haveused both Cox-regression and PAM to correlate expression differenceswith graft outcome with good success. Both methods yield significantresults in Kaplan-Meier survival analysis although at the initial 2-yearfollow-up genes identified by PAM also yield greater significance. (FIG.2—Kaplan-Meier survival analysis for graft loss (red) and no-loss (blue.The genes include ICAM5-FIG. 2A; (p=0.007), IL6R; FIG. 2B; (p=0.003),STAT1; FIG. 2C; (p=0.036), and STAT6; FIG. 2D; (p=0.020)).

The gene signature is dominated by increased expression of cell adhesiongenes, selected cytokines, B-cell genes, representatives in the STATsignaling pathway and several immune response genes including multiplerepresentatives of both class I and class II HLA genes.

Representative genes include those from HLA class I (HLA-F, HLA-G), HLAclass II (HLA-DRB1, HLA-DRB5, HLA-DRB4), signal transducers (STAT1,STAT6), immunoglobulin genes (IGKC, IGHG3), and 2 interferon gammainduced genes (ICAM5, IL6R).

A similar approach was used to identify graft-loss markers in wholeblood samples. The list of the most highly-predictive significant genesin blood is summarized in Table 4, including the Kaplan-Meier survivalsignificance score.

TABLE 4 Fold Unigene Loss/ Symbol Gene ID No-loss p-value HIST1H2BCHistone 1, H2bc Hs.356901 −3.46 0.00018 IGHG3 Ig heavy constant gamma 3(G3m marker) Hs.413826 4.14 0.00134 AHSA2 Activator of heat shock ATPaseHs.122440 2.91 0.00041 TNFRSF10D TNF receptor superfamily 10b Hs.129844−2.55 0.00010 MAPK9 Mitogen-activated protein kinase 9 Hs.348446 8.140.00444 IFNAR2 Interferon (alpha, beta and omega) receptor 2 Hs.86958−2.37 0.01760 TM4SF9 Transmembrane 4 superfamily member 9 Hs.8037 −15.290.00580 MIF Macrophage migration inhibitory factor Hs.407995 −2.310.00674 SCYE1 Small inducible cytokine (Monocyte-activating) Hs.1056562.51 0.00154 MAPK1 Mitogen-activated protein kinase 1 Hs.324473 −2.320.00019 TGFBR3 TGFb receptor III (betaglycan) Hs.342874 −2.94 0.00318IGKC Immunoglobulin kappa constant Hs.377975 2.35 0.00290 IL1R2Interleukin 1 receptor, type II Hs.25333 −4.06 0.01762 IGLImmunoglobulin lambda light chain 3.04 0.02093

The Kaplan-Meier survival curves for 8 of these genes are illustrated inFIG. 3. The genes in FIG. 3 include A) AHSA2, B) IGHG1, C) IFNAR2, D)IGKC, E) HIST1H2BC, F) IL1R2, G) MAPK1, and H) MAPK9.

The functional composition of genes associated with acute rejection,predicted by analysis of Gene Ontology annotations, is summarized inTable 5.

TABLE 5 Genes on EASE Fisher Gene Category Genes Array Score Exactdefense response 105 747 7.15E−12 3.35E−12 response to stimulus/ 1521482 0.00000108 7.24E−07 acute phase response apoptosis 50 3610.00000772 3.63E−06 cell cycle 71 597 0.0000174 9.84E−06 cellproliferation 96 899 0.0000403 0.0000256 protein metabolism 176 19410.000228 0.000172 antigen presentation 9 29 0.000707 0.000123 cellgrowth and/or 244 2887 0.000766 0.000623 maintenance phosphorylation 53512 0.00539 0.00353 protein modification 84 902 0.00775 0.00545hemopoiesis 10 53 0.0116 0.00374 DNA replication 17 122 0.0125 0.00571B-cell activation 6 22 0.0171 0.00356

The full list of known genes (in ranked order) in whole blood that arepredictive of graft loss following acute rejection is summarized inTable 1. Of the 81 cDNA clones identified to have the highest predictivepower, 62 are of known function or assigned unique Unigene Cluster IDs.Similarly, the list of known genes identified in renal biopsiespredictive of graft loss following acute rejection is summarized inTable 2 (including 30 unique genes of known function from the 50 cDNAassociated clones).

IV. Generation of a Transplant Custom Expression Chip TxChip

We have compiled the gene lists described in this document for AR andgraft loss along with other expression-based markers identified to beassociated with clinical outcomes and severity of:

1. Acute rejection—including markers associated with graft loss and/orrate of recovery of renal function following AR (Table 3);

2. Chronic allograft injury (chronic rejection) in blood (Table 3);

3. Immunosuppressive drug toxicity or adverse side effects includingdrug-induced hypertension (Table 3);

4. Age or body mass index associated genes that correlate with renalpathology or account for differences in recipient age-related graftacceptance (Table 3);

5. Immune tolerance markers in whole blood (Table 3);

6. Control genes for assessing sample quality (3′- to 5′-bias in probelocation), sampling error in biopsy-based studies, cell surface markers,and normalizing genes for calibrating hybridization results;

7. Genes found in literature surveys with immune modulatory roles thatmay play a role in transplant outcomes (Table 3) to produce the list fora representative array having probes to genes listed in Table 3.

A. Test of Expression Uniformity Across a Pilot Study of Renal Biopsies.

In the identification of the gene markers described in this inventiondisclosure, we compared the expression across a set of 67 renal biopsiesdescribed in detail by our laboratory. A subset of the biopsy-generatedgene expression markers was used clustered to compare expressionprofiles in patients with confirmed cases of DT, CAN, AR and nosignificant abnormality (Normal). These patients were on two verydifferent immunosuppressant regimes, either steroid-based orsteroid-free (clinical regiment previously described in (Sarwal et al.,Transplantation (2001) 72:13-21) and Sarwal et al., Transplantation(2003) 76:1331-9).

FIG. 4 illustrates that the gene expression is generallyuniform/consistent across the full clinical groups analyzed as the geneexpression levels segregate well within patient groups. Further, withineach group (DT, CAN, AR or Normal) expression levels of these markergenes are independent of immunosuppression use.

The 479 gene list of Table 3 comprises design and specification for acustomized thematic Transplant Chip (TxChip V1) and full-length mRNAsequences for these genes are listed in Table 3. The gene listing iscross-indexed to the studies listed above. We observe a modest overlapin the list of informative genes. For example, expression levels of IGHMpositively correlate with acute rejection risk and negatively correlatewith immune tolerance. An advantage of having the full compilation ofgenes on a common platform is that new discoveries like this can be madein future studies.

It is evident that subject invention provides a convenient and effectiveway of determining whether a graft in a subject will survive, e.g.,following acute rejection. As such, the subject invention provides anumber of distinct benefits, including the ability to identifyclinically relevant AR groups with differing therapeutic responses andprognosis, and allow for individualized treatment and monitoring. Assuch, the subject invention represents a significant contribution to theart.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of evaluating graft survival in asubject gene expression levels of at least two genes in a sample from atransplant recipient, said method comprising: providing a sample from atransplant recipient; and assessingmeasuring an amount of expression ofat least two genes in athe sample from said subject to evaluate graftsurvival in said subject, wherein said at least two genes comprisesHIST1H2BHIST1H2BC and IGHG3.
 2. The method according to claim 1, whereinsaid expression of at least two genes is assessed by assaying saidsample for a nucleic acid transcript of said gene.
 3. The methodaccording to claim 1, wherein said expression of at least two genes isassessed by assaying said sample for an expression product of said gene.4. The method according to any of claim 1, wherein said sample is ablood sample.
 5. The method according to claim 4, wherein said bloodsample is a peripheral blood sample.
 6. The method according to claim 1,wherein said sample is a tissue biopsy sample.
 7. A method according toclaim 1, wherein the method comprises: obtaining an expression profilefor a sample from said subject.
 8. The method according to claim 7,wherein said expression profile is compared to a reference expressionprofile.
 9. The method according to claim 8 7, wherein said expressionprofile is a nucleic acid expression profile.
 10. The method accordingto claim 8 7, wherein said expression profile comprises expressionmeasurements for at least 5 different genes.
 11. The method according toclaim 8 7, wherein said expression profile is determined using amicroarray.
 12. The method according to claim 11, wherein saidmicroarray is a genomic array.
 13. A method of managingpost-transplantation therapy in a subject, said method treating atransplant recipient comprising: (a) evaluating determining that atransplant recipient has a graft survival in said subject by a methodaccording to claim 1; and phenotype by evaluating results previouslyobtained from a quantitative determination of nucleic acid expressionlevels of at least two genes in a sample from the transplant recipient,and treating said transplant recipient by maintaining a currenttherapeutic regimen; or (b) determining a post-transplantation therapyprotocol based on said evaluation step (a); that a transplant recipienthas a graft loss phenotype by evaluating results previously obtainedfrom a quantitative determination of nucleic acid expression levels ofat least two genes in a sample from the transplant recipient, andtreating said transplant recipient by increasing or decreasing atherapeutic regimen; wherein, said evaluating comprises comparing saidresults to a reference nucleic acid expression profile comprising saidat least two genes; and to manage post-transplantation therapy in saidsubjectwherein said at least two genes comprises HIST1H2BC and IGHG3.14. The method according to claim 13, wherein said subject is a human.15. The method according to claim 1, wherein said at least two genesfurther comprises one or more genes selected from: AHSA2, TNFRSF10D,MAPK9, IFNAR2, TM4SF9, MIF, SCYE1, MAPK1, TGFBR3, IGKC, IL1R2 and IGL.16. The method of claim 7, wherein said expression profile comprisesexpression measurements for at least ten different genes.
 17. A methodof assaying gene expression in a blood sample from a graft recipient,the method comprising: a) receiving a sample of blood from a patientthat has received a graft; and b) assaying the expression of at leasttwo genes in the blood sample, wherein said at least two genes comprisesHIST1H2BC and IGHG3.
 18. The method according to claim 13, wherein thetherapeutic regimen is an immunosuppressive therapy.
 19. The methodaccording to claim 13, comprising: determining that the transplantrecipient has a graft loss phenotype that is calcineurin-inhibitor drugnephrotoxicity (DT); and decreasing an immunosuppressive therapy. 20.The method according to claim 13, comprising: (i) determining that thetransplant recipient has a graft loss phenotype that is chronicallograft nephropathy (CAN); and (ii) increasing an immunosuppressivetherapy, or changing an immunosuppressive therapy by administering adifferent immunosuppressive drug.
 21. The method according to claim 1,further comprising measuring an amount of expression of control genes inthe sample.